The present invention relates to the isolation of genes which encode enzymes for the biosynthesis of xcex1-amylase inhibitors, so-called pseudo-oligosaccharides. The genes concerned are, in particular, genes from the Streptomycetes strain Streptomyces glaucescens GLA.O (DSM 40716). In addition, this present patent describes the use of these genes for producing acarbose and homologous substances with the aid of Streptomyces glaucescens GLA.O, the heterologous expression of these genes in other strains which produce pseudo-oligosaccharides (e.g. Actinoplanes sp SE50/100) for the purpose of increasing and stabilizing production, and also their heterologous expression in other microorganisms such as E. coli, Bacillus subtilis, Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella and Streptoporangium strains, Streptomyces hygroscopicus var. limoneus and Streptomyces glaucescens, and also biotechnologically relevant fungi (e.g. Aspergillus niger and Penicillium chrysogenum) and yeasts (e.g. Saccharomyces cerevisiae). The invention also relates to homologous genes in other microorganisms and to methods for isolating them.
Streptomyces glaucescens GLA.O produces the two antibiotics hydroxystreptomycin (Hxc3xctter (1967) Systematik der Streptomyceten (Taxonomy of the Streptomycetes). Basel, Karger Verlag) and tetracenomycin (Weber et al. (1979) Arch. Microbiol. 121: 111-116). It is known that streptomycetes are able to synthesize structurally varied natural products. However, the conditions under which these compounds are produced are frequently unknown, or else the substances are only produced in very small quantities and not detected.
The xcex1-amylase inhibitor acarbose has been isolated from a variety of Actinoplanes strains (SE50, SE82 and SE18) (Schmidt et al. (1977) Naturwissenschaften 64: 535-536). This active substance was discovered in association with screening for xcex1-amylase inhibitors from organisms of the genera Actinoplanes, Ampullariella and Streptosporangium. Acarbose is pseudotetrasaccharide which is composed of an unusual unsaturated cyclitol unit to which an amino sugar, i.e. 4,6-dideoxy-4-amino-D-glucopyranose, is bonded. Additional xcex1-1,4-glycosidically linked D-glucopyranose units can be bonded to the amino sugar. Thus, acarbose, for example, contains two further molecules of D-glucose. The producing strain synthesizes a mixture of pseudo-oligosaccharide products which possess sugar side chains of different lengths (Schmidt et al. (1977) Naturwissenschaften 64: 535-536). The acarbose cyclitol residue is identical to the compound valienamine, which is a component of the antibiotic validamycin A (Iwasa et al. (1979) J. Antibiot. 32: 595-602) from Streptomyces hygroscopicus var. limoneus. 
Acarbose can be produced by fermentation using an Actinoplanes strain and has achieved great economic importance as a therapeutic agent for diabetics. While Actinoplanes synthesizes a mixture of xcex1-amylase inhibitor products, it is only the compound having the relative molecular weight of 645.5 (acarviosin containing 2 glucose units (Truscheit (1984) VIIIth International Symposium on Medicinal Chemistry, Proc. Vol. 1. Swedish Academy of Pharmaceutical Sciences, Stockholm, Sweden), which is employed under the generic name of acarbose. The fermentation conditions are selected to ensure that acarbose is the main product of the fermentation. Alternatives are to use particular selectants and strains in which acarbose is formed as the main product or to employ purification processes for achieving selective isolation (Truscheit (1984) VIIIth International Symposium on Medicinal Chemistry, Proc. Vol. 1. Swedish Academy of Pharmaceutical Sciences, Stockholm, Sweden). It is also possible to transform the product mixture chemically in order, finally, to obtain the desired product acarbose.
In contrast to the genus Streptomyces, the genus Actinoplanes has not so far been investigated intensively from the genetic point of view. Methods which were established for the genus Streptomyces are not transferable, or are not always transferable, to the genus Actinoplanes. In order to use molecular biological methods to optimize acarbose production in a purposeful manner, the genes for acarbose biosynthesis have to be isolated and characterized. In this context, the possibility suggests itself of attempting to set up a host/vector system for Actinoplanes sp. However, this is very tedious and elaborate owing to the fact that studies on Actinoplanes have been relatively superficial.
The invention described in the present patent application achieves the object of cloning the biosynthesis genes for acarbose and homologous pseudo-oligosaccharides, with these genes being cloned from Streptomyces glaucescens GLA.O, which is a streptomycete which has been thoroughly investigated genetically (Crameri et al. (1983) J. Gen. Microbiol. 129: 519-527; Hintermann et al. (1984) Mol. Gen. Genet. 196: 513-520; Motamedi and Hutchinson (1987) PNAS USA 84: 4445-4449; Geistlich et al. (1989) Mol. Microbiol. 3: 1061-1069) and which, surprisingly, is an acarbose producer. In starch-containing medium, Streptomyces glaucescens GLA.O produces pseudo-oligosaccharides having the molecular weights 645, 807 and 970.
Part of the subject matter of the invention is, therefore, the isolation of the corresponding biosynthesis genes from Streptomyces glaucescens GLA.O and their use for isolating the adjoining DNA regions in order to complete the gene cluster of said biosynthesis genes.
The isolation of the genes for biosynthesizing pseudo-oligosaccharides, and the characterization of these genes, are of great importance for achieving a better understanding of the biosynthesis of the pseudo-oligosaccharides and its regulation. This knowledge can then be used to increase the productivity of the Streptomyces glaucescens GLA.O strain with regard to acarbose production by means of established classical and molecular biological methods. In addition to this, the entire gene cluster which encodes the synthesis of the pseudo-oligosaccharides, or individual genes from this gene cluster, can also be expressed in other biotechnologically relevant microorganisms in order to achieve a further increase in, or a simplification of, the preparation of pseudo-oligosaccharides such as acarbose. Specific modification of the biosynthesis genes can also be used to prepare a strain which exclusively produces acarbose having a molecular weight of 645. Since the genes for biosynthesizing antibiotics are always present in clusters and are often very strongly conserved (Stockmann and Piepersberg (1992) FEMS Microbiol. Letters 90: 185-190; Malpartida et al. (1987) Nature 314:642-644), the Streptomyces glaucescens GLA.O genes can also be used as a probe for isolating the acarbose-encoding genes from Actinoplanes sp., for example. The expression of regulatory genes, or of genes which encode limiting steps in the biosynthesis, can result in productivity in Streptomyces glaucescens GLA.O, Actinoplanes sp. or corresponding producer strains being increased. An increase in productivity can also be achieved by switching off (knocking out or mutagenizing) those acarbose biosynthesis genes which have an inhibitory effect in the biosynthesis.
One possible strategy for cloning antibiotic biosynthesis genes which have not previously been isolated is that of using gene-specific probes (Stockmann and Piepersberg (1992) FEMS Microbiol. Letters 90: 185-190; Malpartida et al. (1987) Nature 314:642-644). These probes can be DNA fragments which are P32-labeled or labeled in some other way; otherwise, the appropriate genes can be amplified directly from the strains to be investigated using degenerate PCR primers and isolated chromosomal DNA as the template.
The latter method has been employed in the present study. Pseudo-oligosaccharides such as acarbose contain a 4,6-deoxyglucose building block as a structural element. The enzyme dTDP-glucose 4,6-dehydratase is known to be involved in the biosynthesis of 4,6-deoxyglucose (Stockmann and Piepersberg (1992) FEMS Microbiol, Letters 90: 185-190). Since deoxysugars are a frequent constituent of natural products and antibiotics, this enzyme may possibly be a means for isolating the corresponding antibiotic biosynthesis genes. Since these genes are always present as clusters, it is sufficient to initially isolate one gene; the isolation and characterization of the adjoining DNA regions can then be undertaken subsequently.
For example a dTDP-glucose 4,6-dehydratase catalyzes a step in the biosynthesis of hydroxystreptomycin in Streptomyces glaucescens GLA.O (Retzlaff et al. (1993) Industrial Microorganisms. Basic and applied molecular genetics ASM, Washington D.C., USA). Further dTDP-glucose 4,6-dehydratases have been isolated from other microorganisms, for example from Streptomyces griseus (Pissowotzki et al. (1991) Mol. Gen. Genet. 231: 113-123), Streptomyces fradiae (Merson-Davies and Cundcliffe (1994) Mol. Microbiol. 13: 349-355) and Streptomyces violaceoruber (Bechthold, et al. (1995) Mol. Gen. Genet. 248: 610-620).
It was consequently possible to deduce the sequences for the PCR primers for amplifying a dTDP-glucose 4,6-dehydratase from the amino acid sequences of already known biosynthesis genes. For this, conserved regions in the protein sequences of these enzymes were selected and the amino acid sequences were translated into a nucleic acid sequence in accordance with the genetic code. The protein sequences were taken from the EMBL and Genbank databases. The following sequences were used: Streptomyces griseus; accession number: X62567 gene: strE (dated Oct. 30, 1993); Streptomyces violaceoruber; accession number: L37334 gene: graE (dated Apr. 10, 1995); Saccharopolyspora etythraea; accession number: L37354 gene: gdh (dated Nov. 9, 1994). A large number of possible primer sequences are obtained as a result of the degeneracy of the genetic code. The fact that streptomycetes usually contain a G or C in the third position of a codon (Wright and Bibb (1992) gene 113: 55-65) reduces the number of primers to be synthesized. These primer mixtures can then be used to carry out a PCR amplification with the DNA from strains to be investigated, with the amplification ideally leading to an amplified DNA fragment. In the case of highly conserved proteins, this fragment is of a predictable length which ensues from the distance between the primers in the nucleic acid sequence of the corresponding gene. However, an experimental mixture of this nature does not inevitably have to result in an amplificate. The primers may be too unspecific and amplify a very large number of fragments; alternatively, no PCR product is obtained if there are no complementary binding sites in the chromosome for the PCR primers which have been prepared.
The investigation of the streptomycete strain Streptomyces glaucescens GLA.O resulted in an amplified DNA fragment (acbD ) which had the expected length of 550 bp. Further investigation showed that, besides containing a dTDP-glucose 4,6-dehydratase gene for biosynthesizing hydroxystreptomycin, this strain surprisingly contains a second dTDP-glucose 4,6-dehydratase gene for biosynthesizing pseudo-oligosaccharides such as acarbose. While the two genes exhibit a high degree of homology, they are only 65% identical at the amino acid level.
The acbD probe (see Example 2 and Table 2A) was used to isolate, from Streptomyces glaucescens GLA.O, a 6.8 kb Pstl DNA fragment which encodes a variety of genes (acbA, acbB, acdC, acbD, acbE and acbF) which are involved in the biosynthesis of the pseudo-oligosaccharides.
Deleting the acbBCD genes (aminotransferase, acbB, dTDP-glucose synthase, acbC, dTDP-glucose 4,6-dehydratase, acbD, see Example 6) resulted in the production of a mutant of Streptomyces glaucescens GLA.O which no longer produces any pseudo-oligosaccharides in the production medium. The involvement of the acbBCD genes in the synthesis of pseudo-oligosaccharides was therefore verified by deleting the corresponding loci.
The two genes, i.e. dTDP-glucose synthase and dTDP-glucose 4,6-dehydratase, ought to be involved in the biosynthesis of the deoxysugar of the pseudo-oligosaccharides, as can be concluded from the function of thoroughly investigated homologous enzymes (see above). The amino-transferase (encoded by the acbB gene) is probably responsible for transferring the amino group either to the sugar residue or to the cyclitol residue. By analyzing the protein sequence of acbB, an amino acid motif was found which is involved in binding pyridoxal phosphate. This motif is typical of class III aminotransferases (EC 2.6.1.11; EC 2.6.1.13; EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.62; EC 2.6.1.64; EC 5.4.3.8). The precise enzymic function of acbB can only be elucidated by further investigation of the biosynthesis of the pseudo-oligosaccharides. acbE encodes a transcription-regulating protein which exhibits a great deal of similarity to DNA-binding proteins which possess a helix-turn-helix motif (e.g. Bacillus subtilis DegA, P37947: Swiss-Prot database). Thus, the transcription activator CcpA from Bacillus subtilis inhibits the formation of xcex1-amylase in the presence of glucose, for example (Henkin et al. (1991) Mol. Microbiol. 5: 575-584). Other representatives of this group are proteins which recognize particular sugar building blocks and are able to exhibit a positive or negative effect on the biosynthesis of metabolic pathways. The biosynthesis of the pseudo-oligosaccharides is also regulated in Streptomyces glaucescens GLA.O. It was only previously possible to demonstrate the synthesis of pseudo-oligosaccharides on starch-containing media. While this method indicated that AcbE might be responsible for regulating pseudo-oligosaccharide synthesis, the precise mechanism is still not known. However, molecular biological methods can now be used to modify the gene specifically in order to obtain an increased rate of pseudo-oligosaccharide biosynthesis. Furthermore, the DNA site at which acbE binds can be identified by means of so-called gel shift assays (Miwa et al. (1994) Microbiology 140: 2576-2575). An increase in the rate at which acarbose is biosynthesized can be achieved after identifying and then modifying the promoters and other regulatory DNA regions which are responsible for the transcription of the pseudo-oligosaccharide genes.
At present, the function of acbF is still not definitely known. The corresponding gene product exhibits homologies with sugar-binding proteins such as the sugar-binding protein from Streptococcus mutans (MsmE; Q00749: Swissprot database), making it probable that it is involved in the biosynthesis of the pseudo-oligosaccharides. The gene product of the acbA gene exhibits homologies with known bacterial ATP-binding proteins (e.g. from Streptomyces peucitus DrrA, P32010: SwissProt database). The AcbA protein possesses the typical ATP/GTP binding motif, i.e. the so-called P loop. These proteins constitute an important component of so-called ABC transporters, which are involved in the active transport of metabolites at biological membranes (Higgins (1995) Cell 82: 693-696). Accordingly, AcbA could be responsible for exporting pseudo-oligosaccharides out of the cell or be involved in importing sugar building blocks for biosynthesizing xcex1-amylase inhibitors such as maltose.
All streptomycete genes for biosynthesizing secondary metabolites which have so far been analyzed are arranged in a cluster. For this reason, it is to be assumed that the acarbose biosynthesis genes according to the application are also arranged in such a gene cluster. The remaining genes which are relevant for pseudo-oligosaccharide biosynthesis can therefore also be isolated by isolating the DNA regions which adjoin the 6.8 kb Pstl DNA fragment according to the invention. As has also already been mentioned above, it is readily possible to isolate homologous gene clusters from microorganisms other than Streptomyces glaucescens GLA.O.
The invention therefore relates to a recombinant DNA molecule which comprises genes for biosynthesizing acarbose and homologous pseudo-oligosaccharides, in particular a recombinant DNA molecule in which individual genes are arranged, with respect to their direction of transcription and order, as depicted in FIG. 3 and/or which exhibits a restriction enzyme cleavage site pattern as depicted in FIG. 3, and, preferably, to a recombinant DNA molecule which
(a) comprises a DNA sequence according to Table 4, or parts thereof;
(b) comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to (a), or parts thereof; or
(c) comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules according to (a) and (b) but which permits the expression of the proteins which can be correspondingly expressed using the DNA molecule according to (a) and (b), or parts thereof.
The invention furthermore relates to a recombinant DNA molecule which comprises the acbA gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 1 to 720 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbB gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 720 to 2006 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbC gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 2268 to 3332 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbD gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 3332 to 4306 according to Table 4, or parts thereof; to a recombinant DNA molecule which comprises the acbE gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 4380 to 5414 according to Table 4, or parts thereof; and to a recombinant DNA molecule which comprises the acbF gene, in particular which is characterized in that it comprises the DNA sequence of nucleotides 5676 to 6854 according to Table 4, or parts thereof.
The invention furthermore relates to oligonucleotide primers for the PCR amplification of a recombinant DNA molecule which is as described above and which comprises genes for biosynthesizing acarbose and homologous pseudo-oligosaccharides, with the primers having, in particular, the sequence according to Table 1.
The invention furthermore relates to a vector which comprises a recombinant DNA molecule which comprises a DNA molecule as described in the penultimate and prepenultimate paragraphs, in particular which is characterized in that the vector is an expression vector and said DNA molecule is linked operatively to a promoter sequence, with the vector preferably being being suitable for expression in host organisms which are selected from the group consisting of E. coli, Bacillus subtilis, Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella and Streptosporangium strains, Streptomyces hygroscopicus var. limoneus, Streptomyces glaucescens and also biotechnologically relevant fungi (e.g. Aspergillus niger, Penicillium chrysogenum) and yeasts (e.g. Saccharomyces cerevisiae), with Streptomyces glaucescens GLA.O or Actinoplanes sp. being very particularly preferred. Since the operative linkage of said DNA molecule to promoter sequences of the vector is only one preferably embodiment of the invention, it is also possible for expression to be achieved using promoter sequences which are endogenous in relation to the DNA molecule, e.g. the promoters which are in each case natural, or the natural promoters which have been mutated with regard to optimizing the acarbose yield. Such natural promoters are part of the DNA molecule according to the invention.
The invention furthermore relates to a vector which comprises a DNA molecule according to the invention for use in a process for eliminating or altering natural acarbose biosynthesis genes in an acarbose-producing microorganism. Such a vector is preferably selected from the group consisting of pGM160 and vectors as described in European Patents EP 0 334 282 and EP 0 158 872.
The invention furthermore relates to a host cell which is transformed with one of the above-described DNA molecules or vectors, in particular characterized in that said host cell is selected from the group consisting of E. coli, Bacillus subtilis, Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella or Streptosporangium strains, Streptomyces hygroscopicus var. limoneus or Streptomyces glaucescens, and also biotechnologically relevant fungi (e.g. Aspergillus niger and Penicillium chrysogenum) and yeasts (e.g. Saccharomyces cerevisiae); it is very particularly preferred for it to be selected from the group consisting of Streptomyces glaucescens GLA.O and Actinoplanes sp.
The invention furthermore relates to a protein mixture which can be obtained by expressing the genes of the recombinant DNA molecule according to the invention, comprising genes for biosynthesizing acarbose and homologous pseudo-oligosaccharides, in particular characterized in that the DNA molecule
(a) comprises a DNA sequence according to Table 4, or parts thereof;
(b) comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to (a) or parts thereof; or
(c) comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules according to (a) and (b) but which permits the expression of the proteins which can correspondingly be expressed using the DNA molecule according to (a) and (b), or parts thereof.
The invention furthermore relates to isolated proteins which can be obtained by expressing the genes which are encoded by the DNA molecule described in the previous paragraph.
The following statements apply to all the individual genes identified within the context of the present invention and have only been brought together for reasons of clarity: the invention furthermore relates to a protein which is encoded by a recombinant DNA molecule as described in the last paragraph but one, in particular characterized in that it comprises the DNA sequence of nucleotides 1 to 720 or 720 to 2006 or 2268 to 3332 or 3332 to 4306 or 4380 to 5414 or 5676 to 6854 according to Table 4 or parts thereof; a protein is very particularly preferred which is encoded by the acbA gene or the acbB gene or the acbC gene or the acbD gene or the acbE gene or the acbF gene, and which comprises the amino acid sequence according to Table 4 or parts thereof.
The invention furthermore relates to a process for obtaining the proteins which were described above as being part of the subject-matter of the invention, which process is characterized in that
(a) the proteins are expressed in a suitable host cell, in particular which is characterized in that said host cell is selected from the group consisting of E. coli, Bacillus subtilis, Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella or Streptosporangium strains, Streptomyces, hygroscopicus var. limoneus or Streptomyces glaucescens, and also biotechnologically relevant fungi (e.g. Aspergillus niger and Penicillium chrysogenum) and yeasts (e.g. Saccharomyces cerevisiae); with the host cell very particularly preferably being selected from the group consisting of Streptomyces glaucescens GLA.O and Actinoplanes sp., and
(b) are isolated.
The invention furthermore relates to a process for preparing acarbose, characterized in that
(a) one or more genes of the recombinant DNA molecule which comprises a DNA sequence according to Table 4 or parts thereof or which comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to Table 4, or parts thereof, or which comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules which have just been described but which permits the expression of the proteins which can be correspondingly expressed using these DNA molecules, or parts thereof, is/are used for expression in a suitable host cell which is selected, in particular, from the same group as in the last paragraph, and
(b) the acarbose is isolated from culture supernatants of said host cell.
The invention furthermore relates to a process for preparing acarbose, characterized in that
(a) one or more genes of the recombinant DNA molecule which comprises a DNA sequence according to Table 4 or parts thereof or which comprises a DNA sequence which is able to hybridize, under stringent conditions, with the DNA molecule according to Table 4, or parts thereof, or which comprises a DNA sequence which, because of the degeneracy of the genetic code, differs from the DNA molecules which have just been described but which permits expression of the proteins which can be correspondingly expressed using the DNA molecules, or parts thereof, are eliminated in an acarbose-producing host cell, in particular Streptomyces glaucescens GLA.O and Actinoplanes sp., and
(b) the acarbose is isolated from said host cell.
In this connection, the elimination of one or more genes can be effected by means of standard molecular biological methods, for example using the above-described vectors (pGM160 and others). A gene to be eliminated could, for example, be the acbE gene, which propably has a regulatory function. Genes could likewise be eliminated with the aim of obtaining pure acarbose as the only fermentation product and no longer obtaining a mixture of homologous pseudo-oligosaccharides (see above). The elimination of said genes is preferably achieved using the vectors which have been described above for this purpose.
The invention furthermore relates to a process for preparing acarbose, characterized in that the processes for preparing acarbose which have been described in the previous two paragraphs are combined with each other, such that, therefore, one or more of said genes is/are expressed artificially and one or more of said genes is/are eliminated.
The invention furthermore relates to a process for altering the gene expression of endogenous acarbose biosynthesis genes by mutating the respective gene promoter in order to obtain improved yields of acarbose. In this context, known methods of homologous recombination can be used to introduce the mutations into the production strain to be improved. These mutations can be transitions, deletions and/or additions. An xe2x80x9cadditionxe2x80x9d can, for example, denote the addition of one single nucleotide or several nucleotides or of one or more DNA sequences which have a positive regulatory effect and which bring about an enhancement of the expression of an endogenous gene for biosynthesizing acarbose. The converse case, i.e. the addition of a DNA sequence which has a negative regulatory effect for repressing an endogenous acarbose biosynthesis gene is also a preferred form of an addition. xe2x80x9cTransitionsxe2x80x9d may, for example, be nucleotide exchanges which reduce or amplify the effect of regulatory elements which act negatively or positively. xe2x80x9cDeletionsxe2x80x9d can be used to remove regulatory elements which act negatively or positively. The endogenous genes of this process are preferably present in Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella or Streptosporangium strains, Streptomyces hygroscopicus var. limoneus or Streptomyces glaucescens; very particularly, they are present in Streptomyces glaucescens GLA.O and Actinoplanes sp.
The invention furthermore relates to the use of Streptomyces GLA.O for obtaining acarbose.
The invention furthermore relates to the use of Streptomyces GLA.O for preparing mutants of this strain by the xe2x80x9cclassical routexe2x80x9d, which mutants make it possible to achieve a more abundant production of acarbose. The methods for preparing improved natural product producers of this nature have been known for a long time and frequently make use of classical steps of mutagenesis and selection.
The invention furthermore relates to a process for completing the gene cluster for biosynthesizing acarbose and homologous polysaccharides according to Table 4, characterized in that
a) hybridization probes which are derived from the DNA molecule according to Table 4 are prepared,
b) these hybridization probes are used for the genomic screening of DNA libraries obtained from Streptomyces glaucescens GLA.O, and
c) the clones which are found are isolated and characterized.
The invention furthermore relates to a process for completing the gene cluster for biosynthesizing acarbose and homologous pseudo-oligosaccharides according to Table 4, characterized in that, proceeding from the recombinant DNA molecule according to Table 4,
a) PCR primers are prepared,
b) these PCR primers are used to accumulate DNA fragments of genomic DNA from Streptomyces glaucescens GLA.O, with these primers being combined with those primers which hybridize from sequences of the vector system employed,
c) the accumulated fragments are isolated and characterized.
The invention furthermore relates to a process for isolating a gene cluster for biosynthesizing acarbose and homologous pseudo-oligosaccharides from acarbose-producing microorganisms other than Streptomyces glaucescens GLA.O, characterized in that, proceeding from the recombinant DNA molecule according to claim 4,
a) hybridization probes are prepared,
b) these hybridization probes are used for the genomic or cDNA screening of DNA libraries which have been obtained from the corresponding microorganism, and
c) the clones which are found are isolated and characterized.
The invention furthermore relates to a process for isolating a gene cluster for biosynthesizing acarbose and homologous pseudo-oligosaccharides from acarbose-producing microorganisms other than Streptomyces glaucescens GLA.O, characterized in that, proceeding from the recombinant DNA molecule according to claim 4,
a) PCR primers are prepared,
b) these PCR primers are used for accumulating DNA fragments of gemonic DNA or cDNA from a corresponding microorganism,
c) the accumulated fragments are isolated and characterized, and
d) where appropriate, employed in a process as described in the previous paragraph.
The described processes for isolating a gene cluster for the biosynthesis of acarbose and homologous pseudo-oligosaccharides from acarbose-producing microorganisms other than Streptomyces glaucescens GLA.O are characterized in that the microorganisms are selected from the group consisting of Actinomycetales, such as Streptomyces, Actinoplanes, Ampullariella and Streptosporangium strains, Streptomyces hygroscopicus var. limoneus and Streptomyces glaucescens, preferably from the group consisting of Streptomyces glaucescens GLA.O and Actinoplanes sp.
The invention furthermore relates to the use of Streptomyces glaucescens GLA.O for isolating acarbose.
The invention will now be explained in more detail with the aid of the examples, tables and figures, without being restricted thereto.
All the plasmid isolations were carried out using a Macherey and Nagel (Dxc3xcren, Germany) isolation kit (Nucleobond(copyright)) in accordance with the manufacturer""s instructions. Molecular biological procedures were carried out in accordance with standard protocols (Sambrock et al. (1989) Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, USA) or in accordance with the instructions of the respective manufacturer. DNA and protein sequences were examined using Genetics Computer Group Software, Version 8 (progams: FastA, TFastA, BlastX, Motifs, GAP and CODONPREFERENCE) and the SwissProt (release 32), EMBL (release 46) and Prosite (release 12.2) databases. The molecular biological manipulation of Streptomyces glaucescens and Actinoplanes (DNA isolation and DNA transformations) were carried out as described in Hopwood et al.: Genetic Manipulation of Streptomyces : A Laboratory Manual. The John Innes Foundation, Norwich, UK, 1985 and Motamedi and Hutchinson: Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of Streptomyces glaucescens. Proc. Natl. Acad. Sci. USA 84:4445-4449 (1987).
In general, hybridizations were performed using the xe2x80x9cNon-radioactive DNA labeling kitxe2x80x9d from Boehringer Mannheim (Cat. No. 1175033). The DNA was visualized using the xe2x80x9cLuminescent Detection Kitxe2x80x9d from Boehringer Mannheim (Cat. No. 1363514). In all the examples given in this patent application, hybridization was carried out under stringent conditions: 68xc2x0 C., 16 h. 5xc3x97SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 1% Blocking Reagent (Boehringer Mannheim). SSC denotes 0.15M NaCl/0.015M sodium citrate. The definition of xe2x80x9cstringent conditionsxe2x80x9d which is given here applies to all aspects of the present invention which refer to xe2x80x9cstringent conditionsxe2x80x9d. In this connection, the manner of achieving this stringency, i.e. the cited hybridization conditions, is not intended to have a limiting effect since the skilled person can select other conditions as well in order to achieve the same stringent conditions, e.g. by means of using other hybridization solutions in combination with other temperatures.
The PCR was carried out under standard conditions using in each case 100 pmol of primer 1 and of primer 2 in 100 xcexcl of reaction mixture
The samples are overlaid with 75 xcexcl of mineral oil and the amplification is carried out using a Perkin Elmer TC1 DNA thermal cyler.
Parameters:
Table 1 lists the sequences of the degenerate primers which should be used for amplifying dTDP-glucose dehydratases from different streptomycetes.
The sequencing was performed by the dideoxy chain termination method of Sanger et al. (PNAS USA, 74: 5463-5467 (1977)). The reactions were carried out using the Auto Read Sequenzing Kit(copyright) from Pharmacia Biotech (Freiburg, Germany) in accordance with the manufacturer""s instructions. An ALF DNA Sequencer(copyright) from Pharmacia Biotech (Freiburg, Germany) was used for separation and detection.
The subsequent cloning of the PCR fragments (Sure Clone Kit(copyright), Pharmacia Biotech, Frieburg) into the E. coli vector pUC 18, and the sequencing of the fragment, provided support for the supposition that the fragment encoded a dTDP-glucose 4,6-dehydratase. However, 2 different genes were isolated which both exhibit high degrees of homology with dTDP-glucose 4,6-dehydratase but are not identical. In that which follows, the PCR fragments are designated acbD and HstrE.
The sequences of the isolated PCR fragments are shown in Table 2A and 2B and the homology comparison of the deduced amino acid sequences of HstrE and acbD is shown in Table 2C. The two proteins exhibit an identity of only 65%.
The cells were grown in R2YENG medium and harvested for the DNA isolation after 30 h. The chromosomal DNA was isolated from S. glaucescens GLA.O as described in Hopwood et al. (1985) Genetic manipulations of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich UK).
A Southern blot analysis was carried out using the S. glaucescens GLA.O producer strain chromosomal DNA, which was digested with Pstl, BgIII and BamHI, using the labeled probes consisting of the acbD and HstrE PCR fragments. The two PCR fragments were labeled with digoxygenin in accordance with the manufacturer""s (Boehringer Mannheim; Mannheim) instructions, and a digest of the Streptomyces glaucescens GLA.O producer strain chromosomal DNA was separated on an agarose gel. The DNA was transferred by capillary transfer to nylon membranes and DNA regions which were homologous with the labeled probes were subsequently visualized following hybridization.
The two genes label different DNA regions (FIG. 1 and FIG. 2), with the fragments which were labeled by HstrE having to be gene fragments from Streptomyces glaucescens GLA.O hydroxystreptomycin biosynthesis. While the DNA sequence is not published, the high degree of homology of the protein sequence deduced from HstrE with StrE (Pissowotzki et al. (1991) Mol. Gen. Genet. 231: 113-123) from Streptomyces griseus N2-3-11 streptomycin biosynthesis (82% identity) and the concordance of the HstrE-labeled DNA fragments (FIG. 1) with the published restriction map of the Streptomyces glaucescens GLA.O hydroxystreptomycin gene cluster (Retzlaff et al. (1993) Industrial Microorganisms. Basic and applied molecular genetics ASM, Washington D.C., USA) permits this conclusion. The fragments which were labeled by the acbD probe (FIG. 2) belong to a DNA region which has not previously been investigated. This region encodes the enzymes for biosynthesizing the Streptomyces glaucescens GLA.O pseudo-oligosaccharides.
Inter alia, the acbD PCR fragment labels a 6.8 kB Pstl DNA fragment (FIG. 2). This DNA fragment was isolated as follows. The region of the gel was excised with a razor blade and the DNA was isolated from the gel using an isolation kit from Pharmacia Biotech and cloned into plasmid pUC19 which had been cut with the restriction enzyme Pstl (plasmid pacb1); this latter plasmid was then transformed into the E. coli strain DH5xcex1. The individual clones were subcultured from these plates and a plasmid DNA isolation was carried out using these clones. A PCR amplification using the above-described primers 1 and 2 (Tab. 1) was carried out using the DNA from these clones (250). In this manner, the appropriate E. coli clone containing the 6.8 kb Pstl fragment was isolated.
The DNA was digested with various restriction enzymes and individual DNA fragments were cloned into pUC19. The DNA sequence of the entire fragment, which is shown in Tab. 4 (SEQ ID NO.: 7), was then determined. The DNA sequence of the 6.8 kb Pstl fragment was only partially confirmed by supplementary sequencing of the opposing strand. Several open reading frames, encoding various proteins, were found (programs: CODONPREFERENCE and BlastX). A total of 6 coding regions was found, i.e. a gene having a high degree of homology with ATP-binding protein, acbA, an aminotransferase acbB, a dTDP-glucose synthase acbC, a dTDP-glucose dehydratase acbD, a regulatory gene having homologies with the Lacl protein family acbE, and a protein having similarities to sugar-binding proteins acbF. The sequences of the acbA and acbF genes were only determined in part. The homologies with other proteins from the databases, and the properties of the putative proteins, are summarized in Tab. 3. FIG. 3 shows, in summary form, a restriction map of the fragment, containing the most important restriction cleavage sites mentioned in the text, and the arrangement of the identified open reading frames.
Evidence that the identified DNA fragment encoded pseudo-oligosaccharide biosynthesis genes was produced as follows. A 3.4 kb gene region (EcoR1/Sstl fragment b, FIG. 3) was replaced with the erythromycin resistance gene (1.6 kb) and cloned, together with flanking DNA regions from the 6.8 kb Pstl fragment (pacb1) into the temperature-sensitive plasmid pGM160. The plasmid was constructed as described in the following: the 2.2 kb EcoR1/HindIII fragment (c, FIG. 3) from plasmid pacb1 was cloned into pGEM7zf (Promega, Madison, Wis., USA; plasmid pacb2), and the 1 kb Sstl fragment from pacb1 (a, FIG. 3) was cloned into pUC19 (plasmid pacb3). A ligation was then carried out using the following fragments. The plasmid pGM160 (Muth et al. (1989) Mol. Gen Genet. 219:341-348) was cut with BamH/HindIII, the plasmid pacb2 was cut with Xbal/BamHI (c, FIG. 3), the plasmid pacb3 was cut with EcoRI/HindIII (a, FIG. 3), and the plasmid plJ4026 (Bibb et al. (1985) Gene 38:215-226) was cut with EcoRI/Xbal in order to isolate the 1.6 kb ermE resistance gene.
The fragments were ligated in a mixture and transformed into E. coli DH5xcex1 and selected on ampicillin. The resulting plasmid, i.e. pacb4, was isolated from E. coli DH5xcex1, tested for its correctness by means of restriction digestion and then transferred by protoplast transformation into S. glaucescens GLA.O. The transformants were selected with thiostrepton at 27xc2x0 C. in R2YENG agar. The transformants were subsequently incubated at the non-permissive temperature of 39xc2x0 C. and integration of the plasmid into the genome by way of homologous recombination thereby instituted (selection with thiostrepton (25 xcexcg/ml) and erythromycin (50 xcexcg/ml)). Under these conditions, the only clones which can grow are those in which the plasmid has become integrated into the genome. The corresponding clones were isolated, caused to sporulate (medium 1, see below) and plated out on erythromycin-containing agar (medium 1). Individual clones were isolated once again from this plate and streaked out on both thiostrepton-containing medium and erythromycin-containing medium. The clones which were erythromycin-resistant but no longer thiostrepton-resistant were analyzed. In these clones, the acbBCD genes had been replaced with ermE. Several clones were examined and the strain S. glaucescens GLA.O xcex94acb was finally selected as the reference strain (erythromycin-resistant, thiostrepton-sensitive) for further investigation.
A further experiment examined whether the corresponding strain still produced acarbose. Some clones were grown and investigated for formation of the xcex1-amylase inhibitor in a bioassay; however, no activity was found. The mutants were subsequently further characterized by means of Southern hybridization. Integration of the ermE gene had taken place at the predicted site. FIG. 4 shows a Southern hybridization which was carried out with the wild type and with the Streptomyces glaucescens GLA.O xcex94acb deletion mutant. The Sstl fragment from pacb3 was used as the probe. The chromosomal DNA was isolated from the wild type and mutant and digested with the enzymes Pstl and Pstl/HindIII. The fragment pattern obtained for the deletion mutant corresponds to the predicted recombination event. The wild type exhibits the unchanged 6.8 kb Pstl fragment, whereas the mutant exhibits a fragment which has been truncated by 1.8 kb (compare lanes 1 and 3, FIG. 4). Integration of the ermE resistance gene additionally introduced an internal HindIII cleavage site into the Pstl fragment (compare lanes 2 and 4, FIG. 4).
Using an enzymic test for detecting starch (TC-Starch, Boehringer-Mannheim, Cat. No. 297748), it was possible to demonstrate that the compound isolated from Streptomyces glaucescens GLA.O inhibits xcex1-amylase. Test principle: starch is cleaved into D-glucose by amyloglucosidase. The glucose is then converted with hexokinase into glucose-6-phosphate and the latter is converted with glucose-6-phosphate dehydrogenase into D-gluconate-6-phosphate. This reaction produces NADPH, whose formation can be determined photometrically. Acarbose inhibits the xcex1-amylase and thereby prevents the formation of D-glucose and ultimately the formation of NADPH as well.
The fermentation was carried out, at 27xc2x0 C. on an orbital shaker at 120 rpm, in 500 ml Erlenmeyer flasks which were fitted with side baffles and which contained 100 ml of medium 2. The fermentation was terminated after 2 or 3 days. The pseudo-oligosaccharides were detected in a plate diffusion test as described in Example 9. No xcex1-amylase inhibitors were produced when medium 3 was used. This means that the production of the pseudo-oligosaccharides is inhibited by glucose. Other sugars, such as maltose and sucrose, or complex sugar sources (malt extract) can also come into consideration for producing pseudo-oligosaccharides using S. glaucescens GLA.O.
A suspension of spores of the strain Mucor miehei was poured into agar (medium 5) (105 spores/ml), and 10 ml of this mixture were in each case poured into Petri dishes. Paper test disks (6 mm diameter) were loaded with 10 xcexcl of acarbose [lacuna] (1 mg/ml) or with a sample from an S. glaucescens culture and laid on the test plates. The plates were then incubated at 37xc2x0 C. Inhibition halos appeared on the starch-containing medium 5. A plate which was prepared with glucose (medium 4) instead of starch was used as a control. On this medium, no inhibition halo formed around the filter disks loaded with active compound.
Protoplasts of the Streptomyces glaucescens strain were isolated as described in Motamedi and Hutchinson ((1987) PNAS USA 84: 4445-4449), and the isolated plasmid DNA was transferred into the cells by means of PEG transformation as explained in Hopwood et al. ((1985) Genetic manipulations of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich UK). The protoplasts were regenerated on R2YENG medium at 30xc2x0 C. (Motamedi and Hutchinson (1987) PNAS USA 84: 4445-4449). After 18 h, the agar plates were overlaid with a thiostrepton-containing solution and incubated at 30xc2x0 C. (final concentration of thiostrepton: 20 xcexcg/ml).
Isolation
The culture broth was separated from the mycelium by filtration. The culture filtrate which has been obtained in this way is then loaded onto an XAD16 column, after which the column is washed with water and the active components are eluted with 30% methanol. The eluate was concentrated down to the aqueous phase and the latter was extracted with ethyl acetate in order to remove lipophilic impurities. The aqueous phase was then concentrated and the active components were further purified in 5% methanol using a biogel P2 column. The individual fractions are collected in a fraction collector. The individual fractions were analyzed by means of the Mucor miehei biotest. Active eluates were rechromatographed, for further purification, in 5% methanol on biogel P2. The material which was isolated in this way was investigated by HPLC and MS.
HPLC
Column: Nucleosil(copyright) 100 C-18
Eluent 0.1% phosphoric acid=A/acetonitrile=B
Gradient: from 0 to 100% B in 15 min
Detection: 215 nm
Flow 2 ml/min
Injection volume: 10-20 xcexcl
Using HPLC, it was not possible to distinguish the pseudo-oligosaccharide preparation from S. glaucescens GLA.O from authentic acarbose. Both the retention time and the UV absorption spectrum of the two components were identical in this eluent system. The pseudo-oligosaccharide mixture was not fractionated under these conditions.
Mass spectroscopic analysis (MS)
The molecular weights and the fragmentation pattern of authentic acarbose and the pseudo-oligosaccharides isolated from Streptomyces glaucescens GLA.O were determined by means of electrospray MS. Analysis of the acarbose which is commercially obtainable from Bayer (Glucobay) gave a mass peak at 645.5 (acarbose). The purified samples from S. glaucescens GLA.O contain a mixture of different pseudo-oligosaccharides whose sugar side chains are of different lengths: 969 (acarbose+2 glucose units), 807 (acarbose+1 glucose unit), 645 (corresponds to authentic acarbose). When acarbose and the compound which is isolated from S. glaucescens GLA.O and which has a molecular weight of 645 are fragmented, the same molecular fragments are formed, i.e.: 145 (4-amino-4,6-deoxyglucose), 303 (Acarviosin) and 465 (303 together with one glucose unit).
Actinoplanes sp. SE50 also produces a mixture of acarbose molecules having sugar side chains of different length (Truscheit (1984) VIIIth International Symposium on Medicinal Chemistry, Proc. Vol 1. Swedish Academy of Pharmaceutical Sciences, Stockholm, Sweden). The length of the sugar side chains can be influenced by the choice of the fermentation parameters and of the substrate in the nutrient solution.
The chromosomal DNA was isolated from the strain Actinoplanes sp. SE50/100 and digested with restriction enzymes (Pstl and BamHI). A Southern hybridization was then carried out using a probe which encompasses the coding region of the dTDP-glucose 4,6-dehydratase acbD from Streptomyces glaucescens GLA.O (fragment d, FIG. 3). The probe hybridizes with distinct bands from Actinoplanes sp. SE50/110 (FIG. 5, lanes 1 and 2). This provides the possibility of isolating the corresponding fragments from Actinoplanes sp. SE50/100 and other strain lines. Whether these DNA regions are in fact involved in the biosynthesis of acarbose remains to be demonstrated in subsequent investigations. Alternatively, the PCR primers 1 and 2 (Tab. 1) could also be used for amplifying the dTDP-glucose 4,6-dehydratase from Actinoplanes sp.
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